OBJECTIVE： To establish a method for the simultaneous determination of ethambutol （EMB） and isoniazid （INH） in children’s plasma. METHODS： After precipitated with acetonitrile， plasma samples were determined by UPLC-MS/MS method. Using lamivudine as internal standard， Waters ACQUITY® UPLC HSS T3 column was adopted with mobile phase consisted of acetonitrile-water （containing 0.05％ formic acid and 15 mmol/L ammonium formate） in gradient mode at the flow rate of 0.2 ml/min. By ESI， positive ion detection was conducted in MRM mode. The monitoring transition ion-pair was m/z 205.2→116.0 for EMB， m/z 138.1→121.1 for INH and m/z 230.2→111.9 for internal standard. RESULTS： The linear range of EMB and INH was 10-5 000 ng/ml and 50-5 000 ng/ml， respectively； the limits of quantitation were 10 and 50 ng/ml； RSDs of inter-day and intra-day were all lower than 15％ and relative recoveries were 98.7％-105.5％. Plasma concentrations of EMB and INH in 4 children with tuberculosis were 94.5-99.7 and 55.1-80.9 ng/ml. CONCLUSIONS： The method is rapid and sensitive， which suitable for children’s plasma concentration monitoring of EMB and INH and pharmacokinetic study.